UTILIZATION OF MICROWAVE ASSISTED EXTRACTS OBTAINED FROM VARIOUS PARTS (WHOLE FRUIT, SEEDS, LEAVES AND ROOTS) OF CITRULLUS COLOCYNTHIS AS HYPOCHOLESTEROLEMIC AGENT IN ALBINO RATS

The study was conducted to investigate the hypolipidemic properties of Microwave assisted extracts (MAE) obtained from different parts (whole fruit, leaves, seeds and roots) of Citrullus colocynthis on hyperlipidemic experimental rats. The trial consisted of 30 male rats that were divided into six groups each having 5 rats whereas, out of these 25 were hyperlipidemic and 5 were normal rats. After the induction of high cholesterol for 15 days, 20 rats were fed with microwave assisted extract of different parts at a concentration of 200mg/kg/B. W for 28 days. The administration of the extracts reported considerable reduction in the lipid profile parameters of the hypercholesterolemic rats. The MAE of seed of Citrullus colocynthis displayed the optimum results showing reduced levels of cholesterol, triglyceride and LDL-c by (10.33%), (22.50%) and (15.70%) while an elevation of (16.75%) in HDL-c content compared to the hypercholesterolemic control. Likewise, the percent increase in the concentrations of CAT and SOD by (6.92%) and (18.47%). The study concluded that the MAE of Citrullus colocynthis showed a positive effect on hypercholesterolemia thus providing therapeutic benefits.


INTRODUCTION
Medicinal plants have been known through centuries, due to its potential properties against different diseases and infections. Most of the time such plants are gigantic source of Antioxidants, Flavonoids, Phenolic compounds and Phytochemicals (Demmig-Adams and Adams, 2002). These plants having medicinal properties have vital role throughout world in treating different ailments. As the synthetic medicines have been known to cause many side effects and less effective upon diseases, so the approach to cure diseases with natural medications has increased tremendously (Nimruzi et al., 2013). Citrullus colocynthis has been used as source of energy and remedy for many diseases since early times.it is commonly known as Colocynth, Bitter cucumber, Bitter melon. It is generally grown in desert regions of Asia and Mediterranean regions. Citrullus colocynthis looks like a vine having hard rind small fruits and leaves on it. Roots of the plant have been used to treat snake poison, urinanry tract infection, jaundice, pains in bones and diseases related to eyes. Leaves of Citrullus colocynthis were helpful in therapy for asthma, diuretics and jaundice. Fruit and seeds of Citrullus colocynthis has anti diuretics, anthelmintic effect (Shahid et al., 2019). It also cures tumor, urinary tract infection, joints inflammation, ulcers and asthma diseases. Extracts obtained from different parts of this plant such as stem, leaves, roots, seeds or fruit have positive effect on different ailments (Qureshi et al.,2010). The extraction of components from different plants has been done by numerous techniques. Conventional and non-conventional techniques are practiced since centuries. Non-conventional methods have many benefits such as environmentally friendly, as it consumes less chemicals, less operational time and yields excellent quality of extract as compared to conventional methods (Uma and Sekar, 2014). Microwave assisted extraction (MAE) is one of the unique methods for extracting materials form plants by the help of microwave rays/energy. Microwave assisted extraction technique as numerous advantages such as increased extraction of components from plants due to quick heating, increase extraction yield and small equipment utilized. Due to immense qualities it has been used to extract organic and organo-metallic components from various plants ( Alupului et al., 2012). By considering advantages of MAE, this study was conducted to work on extracts obtained from different parts (whole fruit, leaves, seeds and roots) of Citrullus colocynthis by microwave assisted extraction.

Plant Material
Various parts of Citrullus colocynthis i.e. roots, leaves, seed, & whole fruit was procured from local market of Lahore. These parts were washed, and air-dried at room temperature. Collected roots, leaves, seeds and whole fruit was dried in oven drier at 50±5 0 C to minimize the moisture content. After that they were grinded to reduce the particle size in order to facilitate extraction.

Preparation of Extract
Extraction of Bitter melon roots, leaves, seeds & whole fruit was carried in an adapted commercial kitchen microwave oven. The maximum output of this oven was 700 W. In the MAE procedure, a 25g aliquot of Bitter melon roots, leaves, seeds & whole fruit powder was individually placed in a 250 ml round bottom flask; 25 ml of distilled water was added to moisturize for 30 min. The flask was connected to a Clevenger apparatus and heated at powers of 150 W for varied The study was conducted to investigate the hypolipidemic properties of Microwave assisted extracts (MAE) obtained from different parts (whole fruit, leaves, seeds and roots) of Citrullus colocynthis on hyperlipidemic experimental rats. The trial consisted of 30 male rats that were divided into six groups each having 5 rats whereas, out of these 25 were hyperlipidemic and 5 were normal rats. After the induction of high cholesterol for 15 days, 20 rats were fed with microwave assisted extract of different parts at a concentration of 200mg/kg/B. W for 28 days. The administration of the extracts reported considerable reduction in the lipid profile parameters of the hypercholesterolemic rats. The MAE of seed of Citrullus colocynthis displayed the optimum results showing reduced levels of cholesterol, triglyceride and LDL-c by (10.33%), (22.50%) and (15.70%) while an elevation of (16.75%) in HDL-c content compared to the hypercholesterolemic control. Likewise, the percent increase in the concentrations of CAT and SOD by (6.92%) and (18.47%). The study concluded that the MAE of Citrullus colocynthis showed a positive effect on hypercholesterolemia thus providing therapeutic benefits. extraction time 1, 5, 10 and 15 minutes respectively. The volatile distillate was eluted out by n-hexane and dried through anhydrous sodium sulphate. The n-hexane was removed under vacuum conditions and the extract was refrigerated prior to analysis (Liu et al., 2013).

Figure 1
Graphical representation of experimental design. Microwave assisted extraction of different parts (roots, leaves, fruits and seeds) and their administration to the rats. Analysis of the blood samples after completion of study interval to verify the effect of each part in lowering of lipid

Experimental Animals
Male rats were purchased from animal house of Institute of Molecular Biology and Biotechnology (IMBB), The University of Lahore, having weight between 200g-250g. The rats were kept for 1 week on basal diet for acclimatization purpose. The environmental conditions were controlled throughout the trial like temperature (23 ± 2 °C) and relative humidity (55 ± 5%) along with 12-h lightdark period.

Induction of Hypercholesterolemia
Experimental hypercholesterolemic diet was prepared using corn oil (10%), corn starch (64.5%), cholesterol (1.5%), protein (10%), cellulose (10%), mineral (3%) and vitamins (1%). Groups were subjected to high cholesterol diet for first 15 days for induction of hypercholesterolemia. This was authenticated by examining their total cholesterol content at 15 th day. Rats were anesthetized by exposure to isoflurane and the blood samples were collected through cardiac puncture (Imran et al., 2018).

Experimental design
30 rats were divided into groups, each comprising of 5 rats in them. Group N0 normal control rats fed with their normal diet, Group C0 was fed with only with high cholesterol diet. Group C1 was administered with high cholesterol diet along with Citrullus colocynthis whole fruit extract. Group C2 was administered with high cholesterol diet along with Citrullus colocynthis leaves extract. Group C3 was administered with high cholesterol diet along with Citrullus colocynthis seeds extract. Group C4 was administered with high cholesterol diet along with Citrullus colocynthis roots extract (Kai et al., 2015).

Effect of extract on hyper cholesterol and safety assessment
Rats was anesthetized by exposure to isoflurane and the blood samples was collected in tubes by cardiac puncture and examined at 0 day (baseline trend), 15 th day (post administration of cholesterol rich diet) and 21 st day post induction of hypercholesterolemia along with administration of respective extracts to validate hypocholesterolemic effect (Imran et al., 2018).

Statistical analysis
Data were expressed as mean ±standard deviation and completely randomized design was conducted with two-way (Serum profiling & Antioxidant indices) ANOVA at a significance level of p<0.05 (Steel et al., 1997).

Total Cholesterol
The statistical analysis demonstrated that there was significant (p<0.05) effect of treatment and time intervals on the cholesterol concentrations of the rats.

Total Triglycerides
The statistical analysis demonstrated that there was significant (p<0.05) effect of treatment and time intervals on the triglyceride content of the hypercholesterolemic rats. The effect of microwave assisted extracts obtained from different parts of Citrullus colocynthis on total triglycerides have been displayed in figure 2. Results for total triglycerides observed in Co (173.72+2.33mg/dL) was found to have reduced in C1(137.96+3.21mg/dL), C2 (139.09+2.10mg/dL), C3 (134.33+1.09mg/dL) and C4 (141.56+3.33mg/dL). The figure displayed a significant percent reduction in C3 (22.50%) as compared to experimental group followed by C1 (20.02%), C2 (19.09%) and C4 (18.18%) at the 28 th day of administration of extracts.

LDL
The Statistical analysis revealed that there was significant (p<0.05) effect on treatments and intervals (0, 14 & 28 days) on LDL levels of experimental rats. The values of MAE from various parts of Citrullus colocynthis and their effect on total LDL levels have been mentioned in table 2. Results for the LDL content displayed peak reduction in treatment group C3 (15.70%) followed by C1 (13.95%), C2 (11.77%) and C4 (11.66%), respectively on the 28 th day of administrating MAE. In comparison to the control group Co (113.76±2.01mg/dL), the highest reduction in LDL concentrations were observed in C3 (95.27±2.48mg/dL) followed by C1(97.89±1.57mg/dL), C2(100.36±1.94mg/dL) and C4(100.49±1.70mg/dL), respectively. Furthermore, the value for LDL level at 0 day was 106.20±1.87mg/dL which reduced to 101.44±1.66mg/dL at 14 th day and to 97.03±1.94mg/dL at 28 th day, respectively.

Superoxide Dismutase (SOD)
The statistical analysis observed significant (p<0.05) effect of variables (time intervals and treatments) on the SOD levels in hypercholesterolemic experimental rats. The results for the effect of MAE on the levels of SOD are mentioned in table 4. The levels of SOD were elevated from (12.66±0.22IU/L) at 0 day, to 13.46±0.11IU/L at 14 th day and to 14.22±0.13IU/L at 28 th day respectively. However, the values observed for Co (11.78±0.18IU/L) were noticed to have elevated in C1 (13.19±0.07IU/L), C2 (13.50±0.18IU/L), C3 (14.45±0.30IU/L), C4 (14.30±0.03IU/L), respectively. The percentage elevation in the treatment groups showed highest increase in C3 (18.47%).